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The complement system, well known for its central role in innate immunity, is currently emerging as an unexpected, cell‐autonomous, orchestrator of normal cell physiology. Specifically, an intracellularly active complement system—the complosome—controls key pathways of normal cell metabolism during immune cell homeostasis and effector function. So far, we know little about the exact structure and localization of intracellular complement components within and among cells. A common scheme, however, is that they operate in crosstalk with other intracellular immune sensors, such as inflammasomes, and that they impact on the activity of key subcellular compartments. Among cell compartments, mitochondria appear to have built a particularly early and strong relationship with the complosome and extracellularly active complement—not surprising in view of the strong impact of the complosome on metabolism. In this review, we will hence summarize the current knowledge about the close complosome–mitochondria relationship and also discuss key questions surrounding this novel research area. Linked Articles This article is part of a themed issue on Canonical and non‐canonical functions of the complement system in health and disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.14/issuetoc

Immunologists have recently realized that there is more to the classic innate immune sensor systems than just mere protection against invading pathogens. It is becoming increasingly clear that such sensors, including the inflammasomes, toll-like receptors, and the complement system, are heavily involved in the regulation of basic cell physiological processes and particularly those of metabolic nature. In fact, their "non-canonical" activities make sense as no system directing immune cell activity can perform such task without the need for energy. Further, many of these ancient immune sensors appeared early and concurrently during evolution, particularly during the developmental leap from the single-cell organisms to multicellularity, and therefore crosstalk heavily with each other. Here, we will review the current knowledge about the emerging cooperation between the major inter-cell communicators, integrins, and the cell-autonomous intracellularly and autocrine-active complement, the complosome, during the regulation of single-cell metabolism. LINKED ARTICLES: This article is part of a themed issue on Canonical and non-canonical functions of the complement system in health and disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.14/issuetoc.

SummaryNext generation sequencing technologies have revolutionized the study of T cell biology, capturing previously unrecognized diversity in cellular states and functions. Pathway analysis is a key analytical stage in the interpretation of such transcriptomic data, providing a powerful method for detecting alterations in important biological processes. Current pathway analysis tools are built on models developed for bulk-RNA sequencing, limiting their effectiveness when applied to more complex single cell RNA-sequencing (scRNA-seq) datasets. We recently developed a sensitive and distribution-free statistical framework for multisample distribution testing, which we implement here in the open-source R package Single Cell Pathway Analysis (SCPA). After demonstrating the effectiveness of SCPA over commonly used methods, we generate a scRNA-seq T cell dataset and characterize pathway activity over early cellular activation and between T cell populations. This revealed unexpected regulatory pathways in T cells, such as an intrinsic type I interferon system regulating T cell survival and a reliance on arachidonic acid metabolism throughout T cell activation. A systems level characterization of pathway activity in T cells across multiple human tissues also revealed alpha defensin expression as a hallmark of bone marrow derived T cells. Overall, our work here provides a widely applicable tool for single cell pathway analysis, and highlights unexpected regulatory mechanisms of T cells using a novel T cell dataset.

While serum-circulating complement destroys invading pathogens, intracellularly active complement, termed the ‘complosome’, functions as a vital orchestrator of cell-metabolic events underlying T cell effector responses. Whether intracellular complement is also non-redundant for the activity of myeloid immune cells is currently unknown. Here, we show that monocytes and macrophages constitutively express complement component (C) 5 and generate autocrine C5a via formation of an intracellular C5 convertase. Cholesterol crystal-sensing by macrophages induced C5aR1 signaling on mitochondrial membranes, which shifted ATP production via reverse electron chain flux towards reactive oxygen species (ROS) generation and anaerobic glycolysis to favor IL-1β production, both at the transcriptional level and processing of pro-IL-1β. Consequently, atherosclerosis-prone mice lacking macrophage-specific C5ar1 had ameliorated cardiovascular disease on a high-cholesterol diet. Conversely, inflammatory gene signatures and IL-1β produced by cells in unstable atherosclerotic plaques of patients were normalized by a specific cell-permeable C5aR1 antagonist. Deficiency of the macrophage cell autonomous C5 system also protected mice from crystal nephropathy mediated by folic acid. These data demonstrate the unexpected intracellular formation of a C5 convertase and identify C5aR1 as a direct modulator of mitochondrial function and inflammatory output from myeloid cells. Together, these findings suggest that the complosome is a contributor to the biologic processes underlying sterile inflammation and indicate that targeting this system could be beneficial in macrophage-dependent diseases, such as atherosclerosis.

Pathway analysis is a key analytical stage in the interpretation of omics data, providing a powerful method for detecting alterations in cellular processes. We recently developed a sensitive and distribution-free statistical framework for multisample distribution testing, which we implement here in the open-source R package single-cell pathway analysis (SCPA). We demonstrate the effectiveness of SCPA over commonly used methods, generate a scRNA-seq T cell dataset, and characterize pathway activity over early cellular activation. This reveals regulatory pathways in T cells, including an intrinsic type I interferon system regulating T cell survival and a reliance on arachidonic acid metabolism throughout T cell activation. A systems-level characterization of pathway activity in T cells across multiple tissues also identifies alpha-defensin expression as a hallmark of bone-marrow-derived T cells. Overall, this work provides a widely applicable tool for single-cell pathway analysis and highlights regulatory mechanisms of T cells.

CD4 T cell-intrinsic engagement of the complement receptor CD46 controls nutrient influx and the metabolic reprogramming events that are essential for both the initiation and contraction of human Th1 responses, characterized by IFN-g and IL-10 production respectively. Here, we demonstrate that CD46 also orchestrates T cell arginine metabolism by upregulating the arginine transporter CAT-1 and, unexpectedly, Arginase 1 (Arg1). Arg1 has been well characterized in macrophages where it is associated with the IL-10 secreting M2 type, but its expression or function in T cells has not been described. Surprisingly, in CD4 T cell, Arg1 seems to restrain IL-10 production and contraction: T cells isolated from four patients with rare Arg1 deficiency mount strong Th1 responses but display significantly increased IL-10 switching and early contraction when compared to healthy control cells. Similarly, Arg1fl/fl CD4-cre+ mice infected with influenza virus are characterized by an enhanced Th1 response that contracts more rapidly, resulting in viral control and significantly reduced lung pathology. Unexpectedly, both Arg1-deficient mouse and human T cells produce normal levels of nitric oxide (NO) and polyamines. Metabolic profiling rather revealed that T cells lacking Arg1 have enhanced glycolysis, reduced TCA-cycle intermediates, and engage an “alternative” glutamine pathway often utilized by cancer cells. Normalization of these metabolic perturbations, through the targeting of specific metabolic enzymes, restored typical Th1 induction/contraction. Overall, these data unveil an unexpected intrinsic role for Arginase 1 as a pacemaker of the Th1 lifecycle, which could be harnessed for the amelioration Th1-driven pathologies.

Heme's interaction with Toll-like receptor 4 (TLR4) does not fully explain the proinflammatory properties of this hemoglobin-derived molecule during intravascular hemolysis. The receptor for advanced glycation end products (RAGE) shares many features with TLR4 such as common ligands and proinflammatory, prothrombotic, and pro-oxidative signaling pathways, prompting us to study its involvement as a heme sensor. Stable RAGE-heme complexes with micromolar affinity were detected as heme-mediated RAGE oligomerization. The heme-binding site was located in the V domain of RAGE. This interaction was Fe3+ -dependent and competitive with carboxymethyllysine, another RAGE ligand. We confirmed a strong basal gene expression of RAGE in mouse lungs. After intraperitoneal heme injection, pulmonary TNF-α, IL1β, and tissue factor gene expression levels increased in WT mice but were significantly lower in their RAGE-/- littermates. This may be related to the lower activation of ERK1/2 and Akt observed in the lungs of heme-treated, RAGE-/- mice. Overall, heme binds to RAGE with micromolar affinity and could promote proinflammatory and prothrombotic signaling in vivo, suggesting that this interaction could be implicated in heme-overload conditions.

The complement system is a powerful and druggable innate immune component of the tumor microenvironment. Nevertheless, it is challenging to elucidate the exact mechanisms by which complement affects tumor growth. In this study, we examined the processes by which the master complement regulator factor H (FH) affects clear cell renal cell carcinoma (ccRCC) and lung cancer, two cancers in which complement overactivation predicts poor prognosis. FH was present in two distinct cellular compartments: the membranous (mb-FH) and intracellular (int-FH) compartments. Int-FH resided in lysosomes and colocalized with C3. In ccRCC and lung adenocarcinoma, FH exerted protumoral action through an intracellular, noncanonical mechanism. FH silencing in ccRCC cell lines resulted in decreased proliferation, due to cell-cycle arrest and increased mortality, and this was associated with increased p53 phosphorylation and NFκB translocation to the nucleus. Moreover, the migration of the FH-silenced cells was reduced, likely due to altered morphology. These effects were cell type–specific because no modifications occurred upon CFH silencing in other FH-expressing cells tested: tubular cells (from which ccRCC originates), endothelial cells (human umbilical vein endothelial cells), and squamous cell lung cancer cells. Consistent with this, in ccRCC and lung adenocarcinoma, but not in lung squamous cell carcinoma, int-FH conferred poor prognosis in patient cohorts. Mb-FH performed its canonical function of complement regulation but had no impact on tumor cell phenotype or patient survival. The discovery of intracellular functions for FH redefines the role of the protein in tumor progression and its use as a prognostic biomarker or potential therapeutic target. See article by Daugan et al., p. 891 (36).

Clear-cell renal cell carcinoma (ccRCC) possesses an unmet medical need, particularly at the metastatic stage, when surgery is ineffective. Complement is a key factor in tissue inflammation, favoring cancer progression through the production of complement component 5a (C5a). However, the activation pathways that generate C5a in tumors remain obscure. By data mining, we identified ccRCC as a cancer type expressing concomitantly high expression of the components that are part of the classical complement pathway. To understand how the complement cascade is activated in ccRCC and impacts patients' clinical outcome, primary tumors from three patient cohorts (n = 106, 154, and 43), ccRCC cell lines, and tumor models in complement-deficient mice were used. High densities of cells producing classical complement pathway components C1q and C4 and the presence of C4 activation fragment deposits in primary tumors correlated with poor prognosis. The in situ orchestrated production of C1q by tumor-associated macrophages (TAM) and C1r, C1s, C4, and C3 by tumor cells associated with IgG deposits, led to C1 complex assembly, and complement activation. Accordingly, mice deficient in C1q, C4, or C3 displayed decreased tumor growth. However, the ccRCC tumors infiltrated with high densities of C1q-producing TAMs exhibited an immunosuppressed microenvironment, characterized by high expression of immune checkpoints (i.e., PD-1, Lag-3, PD-L1, and PD-L2). Our data have identified the classical complement pathway as a key inflammatory mechanism activated by the cooperation between tumor cells and TAMs, favoring cancer progression, and highlight potential therapeutic targets to restore an efficient immune reaction to cancer.

Intravascular hemolysis is a hallmark of a large spectrum of diseases, including the sickle cell disease (SCD), and is characterized by liberation of red blood cell (RBC) degradation products in the circulation. Released Hb, heme, RBC fragments and microvesicles (MVs) exert pro-inflammatory, pro-oxidative and cytotoxic effects and contribute to vascular and tissue damage. The innate immune complement system not only contributes to the RBC lysis, but it is also itself activated by heme, RBC MVs and the hypoxia-altered endothelium, amplifying thus the cell and tissue damage. This review focuses on the implication of the complement system in hemolysis and hemolysis-mediated injuries in SCD and in cases of delayed hemolytic transfusion reactions (DHTR). We summarize the evidences for presence of biomarkers of complement activation in patients with SCD and the mechanisms of complement activation in DHTR. We discuss the role of antibodies-dependent activation of the classical complement pathway as well as the heme-dependent activation of the alternative pathway. Finally, we describe the available evidences for the efficacy of therapeutic blockade of complement in cases of DHTR. In conclusion, complement blockade is holding promises but future prospective studies are required to introduce Eculizumab or another upcoming complement therapeutic for DHTR and even in SCD.

International audience; Therapeutic intravenous immunoglobulin preparations (IVIg) are used for treatment of wide range of auto-immune and inflammatory diseases. Versatile mechanisms have been reported to contribute to the im-munomodulatory effects of IVIg. Here we demonstrate that IVIg has a strong potential to inhibit pro-in-flammatory effect of extracellular heme. Indeed, the presence of immunoglobulins reduced the potential of heme to activate the complement system on the surface of human endothelial cells. Since extracellular heme is considered as one of the principal pathogenic factors in hemolytic disorders, its therapeutic scavenging by IVIg may have significant clinical repercussions.

Recent workdefined an unexpected and critical role for intracellular/autocrine active complement in human Th1 biology. Specifically, autocrine stimulation of CD46via TCR-induced C3b generation is needed for IFN-γ production by human CD4+ T cells. Thus, CD46-deficient patients are unable to generate normal Th1 responses. Analysis of the CD46-driven gene signature in Th1 cells revealed CD109 as a direct target of CD46. Indeed, T cells from CD46 deficient patients and from healthy donors in which CD46 expression was ablated via CRISPR-Cas9 technology failed to upregulate CD109. CD109 is aGPI-linked surface protein, mostly expressed on non-immune cells, and belongs to the complement C3 protein family. Though CD109 has been reported as a major negative regulator of TGF-β receptor signaling, direct functional activity for of CD109 on T cells remains unexplored. Knock-out of CD109 in human CD4+ T cells with CRISPR-Cas9 technology induced uncontrolled Th1 and Th17 activation upon in vitro stimulation under non-skewing conditions. Similarly, CD4+T cells from Cd109−/− mice displayed augmented Th1 and Th17 in vitro responses and caused significantly more tissue pathology and weight loss in a T cell-transfer colitis model. Surprisingly, on T cells, CD109 does not restrain IFN-γ and/or IL-17 induction via modulation of TGF-β receptor activity. Instead, CD109 engages with a non-canonical costimulatory molecule and controls stemness- and metabolism-related signaling pathways. Together, these data suggest that the complement-related protein CD109 serves as an important and novel molecular switch on CD4+ T-cells, where it regulates the balance between Th1 and Th17-induction pathways.

International audience; The complement system is an efficient anti-microbial effector mechanism. On the other hand abnormal complement activation is involved in the pathogenesis of multiple inflammatory and hemolytic diseases. As general inhibition of the complement system may jeopardize patient health due to increased susceptibility to infections, the development of pathway-specific complement therapeutics has been a long-lasting goal over the last decades. In particular, pathogen mimicry has been considered as a promising approach for the design of selective anti-complement drugs. The C-terminal domain of staphylococcal superantigen-like protein 7 (SSL7), a protein secreted by Staphylococcus aureus, was recently found to be a specific inhibitor of the terminal pathway of the complement system, providing selective inhibition of cell lysis mediated by the membrane attack complex (MAC). We describe here the selection by phage display of a humanized single-domain antibody (sdAb) mimicking the C-terminal domain of SSL7. The antibody, called sdAb_E4, binds complement C5 with an affinity in the low micromolar range. Furthermore, sdAb_E4 induces selective inhibition of MAC-mediated lysis, allowing inhibition of red blood cell hemolysis and inhibition of complement deposition on apopto-necrotic cells, while maintaining efficient bactericidal activity of the complement terminal pathway. Finally, we present preliminary results indicating that sdAb_E4 may also be efficient in inhibiting hemolysis of erythrocytes from patients with paroxysmal nocturnal hemoglobinuria. Our data provide a proof of concept for the design of a selective MAC inhibitor capable of retaining complement bacteriolytic activity and this study opens up promising perspectives for the development of an sdAb_E4-derived therapeutics with application in the treatment of complement-mediated hemolytic disorders.

Introduction Le syndrome hemolytique et uremique atypique (SHUa) est une microangiopathie thrombotique renale caracterisee par une hemolyse mecanique et une suractivation du complement. Il est demontre que l’heme, derive de l’hemolyse, lie C3a, amplifiant ainsi l’activation du complement. Les mecanismes d’action du complexe heme-C3a sont inconnus. Une interaction du C3a avec le Recepteur aux produits de glycation avancee (RAGE) est rapportee. RAGE, recepteur multi-ligands ubiquitaire, est implique dans differentes pathologies a tropisme renal (nephropathie diabetique, lupique, polykystose) mais aucun role dans le SHUa n’est decrit a ce jour. Notre objectif etait ainsi d’analyser l’interaction C3a-RAGE en conditions hemolytiques. Patients/Materiels et methodes Les interactions entre RAGE, l’heme et C3a etaient etudiees en resonance plasmonique de surface (SPR), spectrophotometrie et chromatographie d’exclusion serique. Pour analyser in vivo l’impact de RAGE en conditions hemolytiques, des souris C57Bl/6 RAGE−/− et leurs controles +/ + littermates recevaient ± une injection intraperitoneale d’heme (50, 100, 200 μmol/kg) avant sacrifice a 16 h. L’expression renale des genes de RAGE et de marqueurs inflammatoires (IL1β, IL6, TNFα) etait etudiee par RTqPCR. Observation/Resultats En SPR, le RAGE recombinant liait la carboxymethyllysine, son principal ligand connu, mais l’interaction RAGE-C3a restait indetectable. A contrario, C3a expose a l’heme liait le RAGE avec une haute affinite. Cette interaction etait donc mediee par l’heme, qui liait RAGE de facon dose-dependante. L’immobilisation des differents domaines de RAGE permettait d’identifier le site de liaison de l’heme sur le domaine V. L’interaction domaine V-heme etait tres affine et specifique, alors que l’heme ne se liait pas aux domaines C1 et C2. Cette liaison etait confirmee en spectroscopie et chromatographie d’exclusion. In vivo, l’injection d’heme a des souris induisait une augmentation dose-dependante de l’expression de RAGE ainsi que l’activation du complement au niveau renal, menant au clivage du C3 et la generation de fragments d’activation du C3 sur l’endothelium glomerulaire. Nous observions egalement une augmentation dose-dependante de l’expression renale des marqueurs de l’inflammation, quantifiee a la condition heme 200 μM/kg autour de × 4, × 200 et × 3,5 pour IL1β, IL6 et TNFα respectivement par rapport au controle (PBS). Les analyses preliminaires montraient une elevation moindre de l’expression de ces genes chez les souris RAGE−/−, dans les memes conditions : × 1,5, × 50 et × 2 respectivement. Discussion/Conclusion Ce travail revele RAGE comme un nouveau recepteur de l’heme. En conditions hemolytiques, les souris RAGE−/− semblent en partie protegees de l’inflammation au niveau renal, ouvrant des perspectives therapeutiques dans les maladies hemolytiques via les molecules anti-RAGE.

In hemolytic diseases, such as sickle cell disease (SCD), intravascular hemolysis results in the release of hemoglobin, heme, and heme-loaded membrane microvesicles in the bloodstream. Intravascular hemolysis is thus associated with inflammation and organ injury. Complement system can be activated by heme in vitro. We investigated the mechanisms by which hemolysis and red blood cell (RBC) degradation products trigger complement activation in vivo. In kidney biopsies of SCD nephropathy patients and a mouse model with SCD, we detected tissue deposits of complement C3 and C5b-9. Moreover, drug-induced intravascular hemolysis or injection of heme or hemoglobin in mice triggered C3 deposition, primarily in kidneys. Renal injury markers (Kim-1, NGAL) were attenuated in C3-/- hemolytic mice. RBC degradation products, such as heme-loaded microvesicles and heme, induced alternative and terminal complement pathway activation in sera and on endothelial surfaces, in contrast to hemoglobin. Heme triggered rapid P selectin, C3aR, and C5aR expression and downregulated CD46 on endothelial cells. Importantly, complement deposition was attenuated in vivo and in vitro by heme scavenger hemopexin. In conclusion, we demonstrate that intravascular hemolysis triggers complement activation in vivo, encouraging further studies on its role in SCD nephropathy. Conversely, heme inhibition using hemopexin may provide a novel therapeutic opportunity to limit complement activation in hemolytic diseases.

Introduction Le syndrome hemolytique et uremique atypique (SHUa) est une microangiopathie thrombotique (MAT) severe, essentiellement renale, caracterisee par une anemie hemolytique, une thrombopenie et une insuffisance renale aigue. Le SHUa est associe a une hyper-activation de la voie alterne du complement (VA), liee a des anomalies genetiques des proteines regulatrices, notamment le facteur H (FH). L’heme, facteur endothelio-toxique libere lors d’une hemolyse, a recemment ete identifie comme activateur de la VA. Cependant, son role ainsi que celui de l’hemolyse dans la physiopathologie du SHUa reste meconnu. Notre objectif a ete d’etudier le role in vivo de ces derniers dans la genese des lesions de MAT dans le SHUa. Patients/Materiels et methodes Pour explorer l’impact de l’hemolyse sur l’apparition de MAT, nous avons injecte de la phenylhydrazine en intra-peritoneale, un agent chimique hemolytique, chez des souris transgeniques deficitaires en FH hepatique versus des souris temoins. Ces souris n’expriment plus que 20 % de FH circulant et sont susceptibles de developper un SHUa apres un evenement declenchant (e.g. serum nephrotoxique). Les parametres hematologiques (plaquettes, hematocrite), histologiques (thrombus) et la fonction renale (uree, proteinurie, hematurie) ont ete analyses a j1, j2 et j4 post-injection. L’expression de marqueurs d’atteinte renale (NGAL, Kim-1), de lesion endotheliale (P-selectine, VCAM, ICAM), de proliferation cellulaire (Ki67), des systemes du complement (FH, C3, C5b9) et de la coagulation (Thrombomoduline, PAI, Facteur tissulaire) ont ete etudies : soit par RT-qPCR, immunohistochimie et immunofluorescence au niveau renal, soit par Western Blot au niveau plasmatique. Observation/Resultats Contrairement aux souris temoins, les souris deficitaires en FH hepatique ont developpes un tableau de MAT des j2 associant insuffisance renale aigue, thrombopenie et apparition de thrombi fibrinoplaquettaires principalement arteriolaires. En parallele, une activation glomerulaire du complement et une agression de l’endothelium sont retrouvees. L’analyse transcriptomique a revele une augmentation des marqueurs de souffrance renale et endotheliale, ainsi qu’une activation de l’hemostase et un defaut de fibrinolyse. Discussion/Conclusion En cas de susceptibilite genetique conduisant a une deregulation du complement, comme dans le SHUa, l’hemolyse massive favorise l’apparition des lesions de MAT. Ces dernieres sont induites par une sur-activation du complement et par une agression endotheliale. Ces resultats ouvrent vers de nouvelles therapeutiques dans le SHUa ciblant l’hemolyse.

Complement is a complex innate immune surveillance system, playing a key role in defense against pathogens and in host homeostasis. The complement system is initiated by conformational changes in recognition molecular complexes upon sensing danger signals. The subsequent cascade of enzymatic reactions is tightly regulated to assure that complement is activated only at specific locations requiring defense against pathogens, thus avoiding host tissue damage. Here, we discuss the recent advances describing the molecular and structural basis of activation and regulation of the complement pathways and their implication on physiology and pathology. This article will review the mechanisms of activation of alternative, classical, and lectin pathways, the formation of C3 and C5 convertases, the action of anaphylatoxins, and the membrane-attack-complex. We will also discuss the importance of structure–function relationships using the example of atypical hemolytic uremic syndrome. Lastly, we will discuss the development and benefits of therapies using complement inhibitors.

Introduction Le syndrome uremique hemolytique atypique (SHUa) traduit des lesions de microangiopathie thrombotique d’expression majoritairement renale. L’identification chez plus de 60 % des patients d’anomalie de regulation de la voie alterne a mis en avant le role majeur du complement. Pour autant, la susceptibilite des cellules endotheliales (CE) renales, alors que l’activation complementaire est systemique, reste incomprise. Materiels et methodes Afin de comprendre le tropisme renal du SHUa, nous avons compare les caracteristiques de differents endotheliums en conditions hemolytiques in vitro et in vivo. Des marqueurs relatifs a l’activation du complement (C3b, iC3b) et a la cyto-protection des CE (facteur H [FH], thrombomoduline [TM], heme oxygenase 1 [HO1]) ont ete etudies dans des cultures de cellules d’origine glomerulaire (culture primaire et lignee), dermique ou de veine ombilicale (HUVEC), ainsi qu’au niveau renal, hepatique et cardiaque dans un modele murin expose a l’heme. Resultats A l’etat basal, le depot et l’expression des differents marqueurs etaient comparables. En revanche, les CE glomerulaires in vitro et l’endothelium glomerulaire in vivo deposaient significativement plus de fragments de C3 apres exposition prolongee a l’heme, par rapport aux HUVEC et aux autres organes testes chez la souris. Nous avons observe sur les CE glomerulaires exposees a l’heme une moindre regulation du complement (plus faible liaison du FH et moindre augmentation de la TM). De plus, contrairement aux autres types cellulaires, l’expression d’HO-1 – enzyme majeure cytoprotectrice degradant l’heme – n’etait pas augmentee sous l’effet de l’heme au sein de l’endothelium glomerulaire aussi bien in vitro qu’in vivo. Seules les HUVEC demontraient une capacite d’adaptation – le depot de C3 induit par l’heme diminuant lors d’une reexposition a ce stimulus – et celle-ci s’averait inhibee en cas de blocage de la fonction HO1. Discussion L’endothelium glomerulaire montre un defaut d’adaptation face a l’hemolyse (moindre synthese d’HO1) et a l’activation du complement (moins de FH, de TM), susceptible de s’exprimer d’autant plus en cas de deregulation sous-jacente de la voie alterne. Conclusion Ainsi, ce changement phenotypique heme-dependant des CE glomerulaires pourrait expliquer l’atteinte endotheliale renale preferentielle dans le SHUa.

Introduction L’heme, en conditions hemolytiques, est un signal de danger qui provoque inflammation et dysfonction cellulaire. Pour reguler ses effets deleteres, il induit l’expression d’heme oxygenase 1 (HO-1), enzyme capable de degrader l’heme et moduler certains genes. Il est responsable de l’hyperactivation du complement a la surface de cellules endotheliales, pouvant contribuer a la pathogenese du syndrome hemolytique et uremique atypique (SHUa). Notre objectif est de caracteriser le role de l’HO-1 dans la protection des cellules endotheliales renales contre l’attaque du complement en situation hemolytique. Materiels et methodes Sur cellules HUVECS et endotheliales glomerulaires, incubees avec des concentrations croissantes d’heme, l’expression d’HO-1 et de regulateurs du complement (MCP, DAF et thrombomoduline) a ete etudiee par RT-qPCR, Western Blot ou cytometrie. Apres rechallenge a l’heme, les depots de C3 (serum normal ou deregule) ont ete etudies en cytometrie. In vivo, des souris C57B6 ont ete injectees par heme ou phenylhydrazine (traitement hemolytique) pour etudier les marquages HO-1, C3 et co-localisations endotheliales dans les reins, cœurs, foies et rates en immuno-histochimie et fluorescence. Resultats Nous mettons en evidence une protection contre l’attaque par le complement sur les HUVECS exprimant l’HO-1, mais pas les cellules endotheliales glomerulaires. L’expression des regulateurs du complement varie avec l’incubation des HUVECs par l’heme. En immuno-histochimie et immunofluorescence, les souris injectees par heme ou phenyl hydrazine ont des depots de C3 glomerulaires et le long de la membrane basale tubulaire. Discussion En conditions hemolytiques in vivo et in vitro, la surexpression d’HO-1 est associee avec des depots de C3 plus faibles. Les depots les plus importants sont intra-glomerulaires ou l’HO-1 n’est pas exprimee. Cet effet protecteur est peut-etre lie a la modulation de l’expression des regulateurs du complement lors d’une surexpression d’HO-1. Conclusion L’absence de protection glomerulaire par l’HO-1 peut expliquer le defaut de protection endotheliale contre l’attaque par le complement, notamment, en cas d’hyperactivation du complement comme dans le SHUa, expliquant potentiellement le tropisme renal du SHUa.

S100 proteins comprise a multigene family of EF-hand calcium binding proteins that engage in multiple functions in response to cellular stress. In one case, the S100B protein has been implicated in oligodendrocyte progenitor cell (OPC) regeneration in response to demyelinating insult. In this example, we report that the mitochondrial ATAD3A protein is a major, high-affinity, and calcium-dependent S100B target protein in OPC. In OPC, ATAD3A is required for cell growth and differentiation. Molecular characterization of the S100B binding domain on ATAD3A by nuclear magnetic resonance (NMR) spectroscopy techniques defined a consensus calcium-dependent S100B binding motif. This S100B binding motif is conserved in several other S100B target proteins, including the p53 protein. Cellular studies using a truncated ATAD3A mutant that is deficient for mitochondrial import revealed that S100B prevents cytoplasmic ATAD3A mutant aggregation and restored its mitochondrial localization. With these results in mind, we propose that S100B could assist the newly synthesized ATAD3A protein, which harbors the consensus S100B binding domain for proper folding and subcellular localization. Such a function for S100B might also help to explain the rescue of nuclear translocation and activation of the temperature-sensitive p53val135 mutant by S100B at nonpermissive temperatures.

International audience; Dynamic interactions between components of the outer (OM) and inner (IM) membranes control a number of critical mitochondrial functions such as channeling of metabolites and coordinated fission and fusion. We identify here the mitochondrial AAA(+) ATPase protein ATAD3A specific to multicellular eukaryotes as a participant in these interactions. The N-terminal domain interacts with the OM. A central transmembrane segment (TMS) anchors the protein in the IM and positions the C-terminal AAA(+) ATPase domain in the matrix. Invalidation studies in Drosophila and in a human steroidogenic cell line showed that ATAD3A is required for normal cell growth and cholesterol channeling at contact sites. Using dominant-negative mutants, including a defective ATP-binding mutant and a truncated 50-amino-acid N-terminus mutant, we showed that ATAD3A regulates dynamic interactions between the mitochondrial OM and IM sensed by the cell fission machinery. The capacity of ATAD3A to impact essential mitochondrial functions and organization suggests that it possesses unique properties in regulating mitochondrial dynamics and cellular functions in multicellular organisms.

ATAD3 is a mitochondrial inner membrane-associated protein that has been predicted to be an ATPase but from which no associated function is known. The topology of ATAD3 in mitochondrial membranes is not clear and subject to controversy. A direct interaction of the N-terminal domain (amino-acids 44-247) with the mtDNA has been described, but the same domain has been reported to be sensitive to limited proteolysis in purified mitochondria. Furthermore, ATAD3 has been found in a large purified nucleoid complex but could not be cross-linked to the nucleoid. To resolve these discrepancies we used two immunological approaches to test whether the N-terminal (amino-acids 40-53) and the C-terminal (amino-acids 572-586) regions of ATAD3 are accessible from the cytosol. Using N-terminal and C-terminal specific anti-peptide antibodies, we carried out back-titration ELISA measurements and immuno-fluorescence analysis on freshly purified human mitochondria. Both approaches showed that the N-terminal region of ATAD3A is accessible to antibodies in purified mitochondria. The N-terminal region of ATAD3A is thus probably in the cytoplasm or in an accessible intermembrane space. On the contrary, the C-terminal region is not accessible to the antibody and is probably located within the matrix. These results demonstrate both that the N-terminal part of ATAD3A is outside the inner membrane and that the C-terminal part is inside the matrix.

Introduction Le syndrome hemolytique uremique atypique (SHUa) constitue un prototype de pathologie liee a une dysregulation de la voie alterne du complement. Pour autant, certains aspects physiopathologiques restent incompris, parmi lesquels le tropisme majoritairement renal des lesions de microangiopathie thrombotique. Notre objectif est d’etudier les capacites de complement- et thrombo-resistances de differentes cellules endotheliales. Materiels et methodes Differents marqueurs relatifs a l’activation du complement (C3b, C5b9), a sa regulation (facteur H (FH), MCP, DAF, CD59) a l’inflammation (ICAM-1) ou a la thrombose (facteur von Willebrand (vWF), thrombomoduline (TM)) ont ete analyses sur des cellules endotheliales primaires glomerulaires humaines (hGEC, 1 donneur) et sur des HUVEC (Human Umbilical Vein Endothelial Cells). Leur expression a ete etudiee par cytometrie de flux ou Elisa, a l’etat basal ou apres incubation 30 minutes en presence d’heme libre, stimulus visant a mimer une hemolyse aigue. Resultats A l’etat basal, le depot de C3 et la formation de C5b9 etaient superieurs a la surface des hGEC par rapport aux HUVEC. Les hGEC produisaient moins de FH mais exprimaient plus fortement MCP, DAF et CD59, ainsi qu’ICAM-1. Apres stimulation par l’heme, le depot de C3 etait plus augmente sur les hGEC que sur les HUVEC et ceci de facon dose-dependante. L’expression de CD55 et de TM diminuaient de facon comparable sur les hGEC et les HUVEC, tandis que la diminution de MCP etait plus forte sur les hGEC. Les HUVEC exprimaient plus de vWF apres stimulation par l’heme par rapport aux hGEC. Discussion et conclusion L’endothelium glomerulaire semble etre une surface naturellement activatrice du complement, malgre la forte expression de regulateurs membranaires. Les hGEC deposent plus de complement sous l’effet de l’heme, ce qui pourrait etre lie a la diminution de l’expression de MCP, de CD55 et la plus faible production de FH. Ces donnees pourraient nous permettre de mieux comprendre le tropisme preferentiel renal du SHUa.